The long term goal of this project is to provide a detailed molecular description of in vivo functions of the Werner syndrome protein (WRN, also known as RECQ3 or RECQL2) in human somatic cells. Specific Aims focus on answering three questions: 1. defining the WRN functional pathway in human somatic cells; 2. identifying in vivo substrates for WRN function in human somatic cells; and 3. determining DNA damage response pathway activation and responsiveness in cells lacking WRN function. This work will test four hypotheses: 1) that WRN acts in somatic cells to resolve aberrant DNA structures or promote the repair of DNA damage that can block, stall or disrupt DNA replication during S-phase; 2) that WRN may play a direct role in the repair of stalled or regressed replication forks; 3) that these resolution/repair functions depend on both catalytic activities of the WRN protein; and 4) that DNA damage response pathways are constitutively activated in human somatic cells that lack WRN, leading to decrements in cell cycle progression, viability and genomic stability. The identification of physiologic roles for the WRN protein in nucleic acid metabolism will provide a detailed understanding of how WRN acts to insure genomic stability, and a model to determine how loss of WRN function promotes the genetic instability, mutagenesis and cellular growth deficits that are central to Werner syndrome pathogenesis.